Fluorescent polymeric materials such as hydrogels and polymeric particles have been attracting attention in many biomedical applications including bio-imaging, optical sensing, tissue engineering and therapy, due to their good biocompatibility, biodegradability, and advanced optical property. This PhD project aims at developing novel autofluorescent protein materials in different configurations with good biocompatibility and biodegradability for bio-imaging applications. Early research focused on the development of autofluorescent protein hydrogels for in vivo bio-imaging application. Glutaraldehyde cross-linked Bovine Serum Albumin (BSA) hydrogel were facilely prepared. Various advanced techniques were employed to characterize the as-prepared material. SEM study clearly revealed its 3-dimentional pore structure, while UV-vis spectra studies, in conjunction with the fluorescence spectra studies including emission, excitation and synchronous scans, indicated that three classes of fluorescent compounds are presumably formed during the gelation process. The autofluorescent hydrogel exhibited high toughness according to the compression and tensile tests. Finally its biocompatibility and biodegradability were demonstrated through extensive in vitro and in vivo studies. More interestingly, the in vivo degradation of autofluorescent hydrogel can be non-invasively tracked using fluorescence images, which provided a convenient way to model in vivo biodegradation of the protein hydrogel from a new perspective. The degradation/diffusion trends predicted by the proposed mathematical model were in good agreement with the time-dependent fluorescence images of mice. Based on the aforementioned autofluorescent concept for BSA system, BSA autofluorescent nanoparticles dispersion and spray-dried microspheres were further fabricated, respectively. Their physical, optical, and biocompatible properties were extensively characterized and evaluated using SEM, FTIR, UV-vis spectra, fluorescence spectra, in vitro cytotoxicity assay, in vivo histological study, and/or Dynamic Light Scattering. The as-synthesized green and red fluorescent nanoparticles dispersion and microspheres were both applied for cell imaging, ascribing to their unique size properties. Also, in vivo degradation processes of these nanoparticles and microspheres in mouse model were also tracked via non-invasive fluorescence imaging and concurrently interpreted by the proposed mathematical model. To further study the degradation mechanism, in vitro degradation of the microspheres by proteinase K were recorded and tracked via Confocal Laser Scanning Microscopy (CLSM), which exhibited two degradation trends based on different concentrations of active enzyme. Microspheres exhibited the swelling of the micro-spherical matrix, accompanying with the decrease of the fluorescent intensity. This phenomena was ascribed to the relatively higher diffusing rate of the enzyme into microspheres matrix than that of the accompanying enzyme-based matrix degradation. A mathematical model was proposed to demonstrate the complexing of microsphere swelling, enzyme diffusion, and diffusion of the liberated fluorophores from enzyme-degraded BSA microspheres matrixes. As another application of the developed autofluorescent protein materials, the as-synthesized autofluorescent BSA nanoparticles have been applied for sensitive heme/hemin detection, and the ultrasensitive sensing performance is ascribed to Photo-induced Electron Transfer (PET) as well as specific interaction between hemin and the fluorescent protein nanoparticles. Overall, this dissertation expands the cutting edge in the design and synthesis of autofluorescent materials with good biocompatibility and biodegradability for various biomedical applications.
